An imaging detection system is designed to achieve quantitative measurement of the concentration of fluorescent immune-chromatographic test strip. Using 365 nm ultraviolet light emitting diode (LED) as the excitation light, we capture the clear image of fluorescent immune-chromatographic test strip by the hand-held zoom microscope camera. Genetic algorithm on the base of Otsu is used to segment the image according to the characteristic of relatively fixed position of the detection line and the quality control line. Then, the background noise is removed by the fluorescence region localization method. The segmented fluorescence image background is filtered out and the gray values of the detection line and the quality control line are calculated. Finally, the eigenvalues of the fluorescence region are calculated to realize the quantitative analysis for the concentration of the fluorescence test strip. The experimental results show that the fluorescence detection system has good repeatability. Five different concentrations of fluorescent test strips are tested for ten times. The variable coefficients of the results are less than 0.5%. The fitting degree of the standard curve reaches 0.99944, and the rapid quantitative detection is realized.